Heterosis in crop plants can have a marked effect on yield improvement. In general, hybrids exhibit increased yields in comparison with non-hybrid varieties and usually give greater return unit for growth factors such as water and fertilizer. Hybrids often offer superior stress tolerance, uniformity in product and maturity and also afford a simple breeding opportunity to combine characteristics or traits that may be difficult to combine in other ways. Hybrid vigor in plants is generally of sufficient magnitude to warrant commercial exploitation. Commercial hybrids are used extensively in many crops including corn, sorghum, sugar beet, sunflower and canola. Despite the many advantages of hybrid vigor, wheat, barley and rice are still grown mainly as inbreeds, primarily due to the lack of economical hybrid seed production methods.
A precondition for the production of hybrid varieties is the directed pollination of a mother line using a selected father line as the pollen donor. In order to produce a sufficient number of seeds, the latter step has to be performed on large-scale and under outdoor conditions. By cultivating a pollenless, i.e. male sterile, mother line and a fertile (pollen-bearing) father line in direct proximity, such a directed pollination is achieved, thereby creating a large number of hybrid seeds that can be directly traced back to the crossing combination of the two parent lines. A precondition particularly for this is the existence of a complete, i.e. if possible, 100% male sterile mother line which cannot pollinate itself.
For this reason various methods were developed in the past, in particular mechanical, chemical and genetic methods for the induction of male sterility of plants. Mechanical methods, such as the removal of the anthers, are only suitable for plant species having large and/or spatially separated sexual organs, such as is the case in corn (Zea mays L.). For the chemical emasculation of plants, substances called gametocides were developed, which have a lethal effect on pollen after application. In this way, hybrid varieties of strict self-pollinators such as wheat and barley could be produced for the first time.
Genetic mechanisms that induce male sterility of plants have been previously described. For instance, male sterile plants basically occur in the mostly aneuploid progeny of wide, i.e. inter-specific or intergeneric crossings. This is partly due to irregularities in the meiosis of the progeny affecting both male and female gametes to the same extent. Additional, systems that are based on single gene defects and only influence the male gametes and pollen have been discovered and further refined. Such systems can be traced back on the one hand to mutations in the nuclear genome (nuclear male sterility, NMS) and on the other hand to gene alterations in the plastom or cytoplasm (cytoplasmic male sterility, CMS).
In most higher plants, cytoplasmic male sterility (CMS) is inherited strictly maternally. CMS is based in principle on the incompatibility of the nucleus and the cytoplasm. With the help of a corresponding father line, not able to overcome the sterility of the maternal cytoplasm (so-called “maintainer”), a homozygous sterile CMS plant may theoretically be produced after repeated back-crossing with the maintainer plant. A complete CMS system, for the production of hybrid seeds of grasses whose vegetative mass is used, thus consists of the following components: (1) the CMS line which bears a sterility-inducing cytoplasm (S), also called sterile mother line; (2) the maintainer line, which bears a normal fertile cytoplasm (N) and which is very similar to the CMS line in other respects; (3) the pollinator line or father line, which has normally fertility and is suitable for combination with the CMS mother line.
A fundamental technical problem for the production of hybrid varieties is the stability of male sterility in the CMS line. This particularly affects the 100% transfer of male sterility to the next generation after crossing and the provision of an environmentally independent phenotype in the form of male sterile plants. Only under these conditions can agronomically optimized and complete male sterile mother plants be generated, which permit heterosis in the form of hybrid varieties to be exploited to its full extent and to realize an additional yield potential.
The Lolium species perennial ryegrass (Lolium perenne L.), annual ryegrass (Lolium multiflorum L.) and hybrid ryegrass (Lolium hybridum L.) are the most important grass species in European food grass culture. For food grasses the specific exploitation of heterosis effects is thought to be a real possibility for substantially increasing yields and for improving further quantitative characteristics such as stress tolerances against biotic and abiotic factors. As the aforementioned Lolium species are cross-pollinators, the opportunity for breeding of synthetics and hybrid varieties presents itself for this purpose.
In order to achieve additional variability as a basis for the selection of new genotypes, the method of polyploidization is used in the breeding of cultured plants. In polyploidization, using mitosis inhibitors such as colchicine during mitosis allows the chromosome set of a cell to be doubled. In the case of Lolium species this leads to the generation of tetraploid forms from originally diploid species (2n=2x=14), which have a double chromosome set (2n=4x=28). Because tetraploids possess other characteristics besides diploids, for the economically relevant Lolium species L. perenne, L. multiflorum and L. hybridum corresponding tetraploid varieties have been cultivated.
For ryegrass species numerous studies point to heterosis and hybrid growth in all valences (including C. A. Foster, Interpopulational and intervarietal hybridization in Lolium perenne breeding, heterosis under noncompetitive conditions, J. Agric. Sci. 1971, 107-130; C. A. Foster, (1973): Interpopulational and intervarietal F1-Hybrids in Lolium perenne: performance in field sward conditions, J. Agric. Sci. 1973, 80, 463-477; I. Rod, Beitrag zu den methodischen Fragen der Heterosiszuchtung bei Futtergrasern, Ber. Arbeitstagung Arbeitsgemeinsch. Saatzuchtleiter Gumpenstein 1965, pages 235-252; I. Rod, Remarks on heterosis with grasses, Heterosis in plant breeding, Proc. 7th Congr. Eucarpia Budapest (1967), pages 227-235; A. J. Wright, A theoretical appraisal of relative merits of 50% hybrid and synthetic, J. Agric. Sci. 79, 1972, pages 245-247). In the past, heterosis effects could be detected particularly after single plant crossings, line crossings and variety crossings (Kobabe, see above).
The breeding method most commonly used at present, namely the production of synthetics or varieties on the basis of clones or populations, was developed for grasses by Frandsen in 1940 (N. H. Frandsen, Some breeding experiments with timothy, Imp. Agric. Bur. Joint Publ. 1940, 3, 80-92). As mentioned above, however, this method only allows a partial use of heterosis. A true food grass hybrid variety using a Lolium line with cytoplasmic male sterility for the complete utilization of heterosis (C. Bothe, see above; G. Kobabe, see above; V. Lein, see above) is not known so far, because no plants with complete male sterility were available and the known CMS sources are unstable.
The systems found or used for Lolium species for the achievement of male sterility differ with respect to their origin and mode of action. Systems with mechanical control for the castration of the plants are ruled out for Lolium species due to their morphology. Chemical methods have not yet been developed for Lolium, while genetic control mechanisms were described previously. Spontaneously generated sources have been reported by Nitzsche (Cytoplasmatische mannliche Sterilitat bei Weidelgras (Lolium ssp.) Z. Pflanzenzucht., Berlin (West) 65, (1971), pages 206-220) for Lolium multiflorum, and, for Lolium perenne, by Gaue (Moglichkeiten der Hybridzuchtung auf ms-Basis bei Lolium perenne L. XIII. Internat. Grasland-Kongreβ, Leipzig 1977, Sektionsvortrag 1-2, pages 491-496; Ergebnisse von Untersuchungen zur Hybridzuchtung bei Lolium perenne Tag.-Ber., Akad. Landwirtsch.-Wiss. DDR, Berlin (1981) 191, pages 119-126). After species and genus crossings male sterile forms also developed for Lolium perenne (F. Wit, Cytoplasmic male sterility in ryegrasses (Lolium ssp.) detected after intergeneric hybridization, Euphytica 1974, 23, 31-38; V. Connoly, Hybrid grasses varieties for the future Farm Food Res. 1978, 9, 6, 131-132; V. Connoly, R. Wright-Turner, Induction of cytoplasmic male-sterility into ryegrass (Lolium perenne), Theor. Appl. Genet. 1984, 68, 449-453). None of these genetic systems could however be stabilized genotypically and phenotypically, so that as yet no functional hybrid system is known for the different ryegrass species.
Although the production of hybrid lines with improved agronomic characteristics is intensively studied, methods available so far for the production of male sterile plants do not lead to completely satisfactory results in many cases. Therefore a strong need for a method for the production of completely male sterile and stable plants that do not show the disadvantages of the prior art is clearly indicated.